Identification of secreted proteins as detection markers for citrus disease

ABSTRACT

Secreted proteins as detection markers for insect vector and graft transmitted citrus disease are described. Method and kits for detecting the secreted proteins are provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 13/829,270, filed on Mar. 14, 2013, which claims benefit of priority to U.S. Provisional Application No. 61/615,760, filed on Mar. 26, 2012, the disclosure of which is hereby incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

Citrus production is one of the most important agricultural economic activities in the United States and around the world. According to the Food and Agricultural Organization (FAO) of the U.N., approximately 47,170 tons of citrus fruits such as oranges, lemons, grapefruits, etc., were produced in 2011, corresponding to US$2.3 billion from sales of fresh fruits and juices around worldwide (FAOSTAT). However, the citrus industry has been experiencing a big threat from phloem-colonizing and insect-transmitted bacterial diseases including Citrus Stubborn Disease (CSD) and Huanglongbing (HLB, also known as citrus greening). Diagnosis of these diseases has been very challenging because of the low titer and uneven distribution of the pathogens in the citrus tree.

The causative bacteria Candidatus Liberibacter (for HLB) and Spiroplasma citri (for CSD) reside exclusively in the phloem of infected trees once they are introduced by the phloem-feeding insect vectors or by grafting. So far, CSD or HLB resistant varieties of citrus have not been found.

The most commonly used pathogen detection methods are PCR based, which requires the presence of bacterial cells or DNA on the tested sample for positive diagnosis. This is been problematic because the low titer and variable distribution of the pathogen within infected trees. Both S. citri and Ca. Liberibacter cells exhibit extremely uneven distributions in infected trees. Very often, the pathogen cells cannot be detected even in symptomatic branches or leaves. Moreover, nucleic acid-based assays require sample preparations, which can be complex, costly and time consuming, especially when numerous samples have to be tested for one tree. So far, the ability to process thousands of samples necessary to track an epidemic using nucleic acid-based methods remains manpower and cost challenging.

Bacteria pathogens secrete numerous proteins to the environment and some of these secreted proteins are essential virulence factors functioning in plant cells. Genes encoding these specialized pathogenesis-related protein secretion apparatus are present in the host plant, but absent from S. citri and Ca. Liberibacter. Bacterial pathogens are injected into plant tissues, i.e., the phloem, by their corresponding insect vectors at the initial stage of infection; therefore, proteins secreted from the general secretion system are readily delivered inside the host cells. This is consistent with the observation that the pathogen cells are often absent from symptomatic tissues. While not being bound by any particular theory, it is believed that the presence of secreted proteins are responsible for the symptom development. Importantly, there are no curative methods once the trees are infected. The success of disease management is largely dependent on early pathogen detection.

BRIEF SUMMARY OF THE INVENTION

One aspect presented herein is a method of detecting citrus stubborn disease (CSD) in a citrus plant. The method includes detecting the presence or absence of a secreted protein from Spiroplasma citri in a sample from the plant, whereby the presence of the secreted protein indicates that the plant has citrus stubborn disease. In some embodiments, the sample or sap is not subjected to protein separation or other methods of isolating, extracting or purifying proteins in the sample prior to the detecting step.

In some embodiments, the secreted protein for detecting CSD is encoded by CAK98563, or is substantially identical to the protein encoded by CAK98563. The CAK98563 protein (SEQ ID NO:1), also referred to as ScCCPP1, is predicted to be a transmembrane lipoprotein. In some embodiments, the secreted protein is encoded by CAK99824, or is substantially identical to the protein encoded by CAK99824. The CAK98563 protein (SEQ ID NO:2) is predicted to be a transmembrane lipoprotein. In some embodiments, the secreted protein is identical or substantially identical to a protein listed in Table 1.

Another aspect presented herein is a method of detecting citrus greening disease (Huanglongbing or HLB) in a citrus plant. The method includes detecting the presence or absence of a secreted protein from Candidatus Liberibacter asiaticus in a sample from the plant, whereby the presence of the secreted protein indicates that the plant has HLB. In some embodiments, the sample or sap is not subjected to protein separation or other methods of isolating, extracting or purifying proteins in the sample prior to the detecting step.

In some embodiments, the secreted protein for detecting HLB is identical or substantially identical to a protein listed in Table 2.

In some embodiments, the citrus plant is not artificially infected or graft-inoculated with a bacterial pathogen.

In some embodiments, the secreted protein is detected by detecting the specific binding of an antibody to the secreted protein. In some instances, the antibody binds to the secreted protein encoded by CAK98563, or to the secreted protein substantially identical to the protein encoded by CAK98563. In some instances, the antibody binds to the secreted protein encoded by CAK99824, or to the secreted protein substantially identical to the protein encoded by CAK99824. In some instances, the antibody binds to a secreted protein that is identical or substantially identical to a protein listed in Table 1. In other instances, the antibody binds to a secreted protein that is identical or substantially identical to a protein listed in Table 2.

In some embodiments, if a plant is determined to have CSD (Spiroplasma citri) or HLB (Candidatus Liberibacter asiaticus , the infected plant is removed and or destroyed.

Another aspect presented herein is a kit for detecting citrus stubborn disease. The kit contains one or more reagent specific for a secreted protein from Spiroplasma citri. In some instances, the reagent is an antibody. In some embodiments, at least one reagent of the kit is an antibody that specifically binds to the secreted protein encoded by CAK98563, or to the secreted protein substantially identical to the protein encoded by CAK98563. In some embodiments, at least one reagent of the kit is an antibody that specifically binds to the secreted protein encoded by CAK99824, or to the secreted protein substantially identical to the protein encoded by CAK99824. In some embodiments, at least one reagent of the kit is an antibody that specifically binds to a secreted protein that is identical or substantially identical to a protein listed in Table 1.

Another aspect presented herein is a kit for detecting citrus greening disease. The kit contains one or more reagent specific for a secreted protein from Candidatus Liberibacter asiaticus. In some instances, the reagent is an antibody. In some embodiments, at least one reagent of the kit is an antibody that specifically binds to a secreted protein that is identical or substantially identical to a protein listed in Table 2.

In some embodiments, the antibody of the kit for CSD or HLB is labeled. In some instances, the antibody has a detectable label (e.g., fluoroscent dye, enzyme, biotin, etc.).

In some embodiments, the antibody of the kit for CSD or HLB is linked to a solid support. Non-limiting examples of a solid support include the surface of an ELISA plate, a glass slide, a coated plate, a bead such as a silica, plastic or derivatized plastic, paramagnetic or non-magnetic metal bead, a polymeric gel or matrix, or a filter, such as a nylon or nitrocellulose.

In some embodiments, the kit for CSD comprises more than one antibody that specifically binds to any one of the proteins (or substantially identical variants thereof) described in Table 1, wherein a first antibody that binds the protein of interest is linked to a solid support and a second antibody that binds the protein of interest is labeled with a detectable moiety.

In some embodiments, the kit for HLB comprises more than one antibody that specifically binds to any one of the proteins (or substantially identical variants thereof) described in Table 2, wherein a first antibody that binds the protein of interest is linked to a solid support and a second antibody that binds the protein of interest is labeled with a detectable moiety.

Also provided are antibodies (optionally isolated) that specifically bind to secreted proteins as described herein. In some embodiments, the antibody specifically binds a secreted protein that is identical or substantially identical to a protein listed in Table 1. In some embodiments, the antibody that specifically binds to the secreted protein (i.e., SEQ ID NO:1) encoded by CAK98563, or to the secreted protein substantially identical to the protein (i.e., SEQ ID NO:1) encoded by CAK98563. In some embodiments, the antibody that specifically binds to the secreted protein encoded by CAK99824 (i.e., SEQ ID NO:2), or to the secreted protein substantially identical to the protein (i.e., SEQ ID NO:2) encoded by CAK99824. In some embodiments, the antibody specifically binds a secreted protein that is identical or substantially identical to a protein listed in Table 2. In some embodiments, the antibody is detectably-labeled. In some embodiments, the antibody linked to a solid support.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show Western blot analysis of ScCCPP1 protein encoded by CAK98563 and spiralin protein in S. citri cells alone and S. citri cells in phloem extracts. FIG. 1A shows that the secreted protein is present in S. citri cells alone and S. citri cells in phloem extracts. FIG. 1B shows that spiralin is present in all samples containing S. citri.

FIG. 2 illustrates Western blot analysis of ScCCPP1 protein in S. citri cells, grafting-inoculated plant tissue and CSD-infected trees from the field.

FIG. 3A illustrates that the anti-ScCCPP1 antibody specifically detects S. citri using leaf petiole imprints. FIG. 3B shows that spiralin protein was not detected in the same samples using the spiralin antibody of FIG. 1B.

FIGS. 4A-4B illustrate one embodiment of the method described herein. FIG. 4A shows an exemplary membrane-based immunoassay or imprinting assay performed on samples harvested from the field. FIG. 4B shows that the imprinting assay is more sensitive than quantitative PCR for detecting CSD.

DETAILED DESCRIPTION OF THE INVENTION I. INTRODUCTION

Methods for serologically diagnosing CSD using S. citri-specific secreted protein or HLB using C. liberibacter-specific secreted protein in a sample are provided. Bacterial pathogens secrete effector proteins into their hosts during infection. These effectors are usually unique for specific pathogen species or even subspecies. It has been discovered that secreted effector proteins can be used as detection markers for diagnosis of bacterial diseases. Antibodies generated to specifically recognize suitable effector proteins can be used to develop serological detection methods such as enzyme-linked immunosorbent assay (ELISA) and imprint detection. The method described herein is particularly efficient for detecting disease in trees where detection of pathogens using nucleic acid-based methods, such as polymerase chain reaction, is challenging due to the uneven distribution of the pathogens in the infected trees. Furthermore, many bacterial pathogens reside in plant transportation systems, i.e. xylem and phloem; therefore, effectors secreted from the pathogens can be dispersed through nutrient and water transportation.

The method described below is advantageous over other detection methods such as nucleic acid-based detections in two ways: 1) it can overcome the problem of erratic distribution and low titer of the pathogens in the host plant; 2) antibody-based serological detection methods allow rapid and economical processing of a large amount of samples in applications like field surveys.

II. DEFINITIONS

As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a plant” includes a plurality of such plants and reference to “the tree” includes reference to one or more trees known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although any methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods and materials are now described.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”

Two nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

The phrase “substantially identical,” used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least 60% sequence identity with a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%. Some embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.

Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10⁻⁵, and most preferably less than about 10⁻²⁰.

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is the only natural codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for one another:

-   1) Alanine (A), Serine (S), Threonine (T); -   2) Aspartic acid (D), Glutamic acid (E); -   3) Asparagine (N), Glutamine (Q); -   4) Arginine (R), Lysine (K); -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g.,     Creighton, Proteins (1984)).

The phrase “specifically binds,” when used in the context of describing a binding relationship of a particular molecule to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated binding assay conditions, the specified binding agent (e.g., an antibody) binds to a particular protein at least two times the background and does not substantially bind in a significant amount to other proteins present in the sample. Specific binding of an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein or a protein but not its similar “sister” proteins. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or in a particular form. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective binding reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.

A “label,” “detectable label,” or “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins that can be made detectable, e.g., by incorporating a radioactive component into the peptide or used to detect antibodies specifically reactive with the peptide. Typically a detectable label is attached to a molecule (e.g., antibody) with defined binding characteristics (e.g., a polypeptide with a known binding specificity), so as to allow the presence of the molecule (and therefore its binding target) to be readily detectable.

The term “plant” includes whole plants, shoot vegetative organs and/or structures (e.g., leaves, stems and tubers), roots, flowers and floral organs (e.g., bracts, sepals, petals, stamens, carpels, anthers), ovules (including egg and central cells), seed (including zygote, embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), seedlings, plant tissue (e.g., vascular tissue, ground tissue, and the like), cells (e.g., guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the methods of the invention includes angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, bryophytes, and multicellular and unicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid, and hemizygous. In some embodiments, the secreted protein is detected in a biological sample from a citrus plant. For example, the biological sample 0can comprise fluid or sap from bark, fruit, a leaf, a leaf petriole, a branch, a twig, or other tissue from an infected or control plant. In some embodiments, the citrus plant is an orange tree, a lemon tree, a lime tree, or a grapefruit tree. In one embodiment, the citrus plant is a navel orange, Valencia orange, sweet orange, mandarin orange, or sour orange. In one embodiment, the citrus plant is a lemon tree. In one embodiment, the citrus plant is a lime tree. In some embodiments, the plant is a relative of a citrus plant, such as orange jasmine, limeberry, and trifoliate orange.

“Antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody or its functional equivalent will be most critical in specificity and affinity of binding. See Paul, Fundamental Immunology.

III. DETAILED DESCRIPTION OF EMBODIMENTS

Herein is provided a serological detection method for monitoring the effectors secreted from the pathogens into the phloem, wherein the pathogen secreted proteins are markers for citrus stubborn disease. The method includes using an antibody to detect a secreted protein of the bacterial pathogen Spiroplasma citri. Because the causal pathogens Candidatus Liberibacter and Spiroplasma citri reside in the phloem of infected trees, secreted proteins are dispersed throughout the tree along with the transportation flow in the host vascular system. Therefore, although the pathogens cells have a restricted and sporadic distribution pattern, the pathogen proteins are not restricted to the infection sites. This allows for robust detection and overcomes the difficulty from the uneven distribution of the pathogen cells in infected trees. Furthermore, direct detection of pathogen-associated patterns are more reliable and highly selectivity, especially compared to detections of host changes, which may not be specific to a particular disease.

Accordingly, in some embodiments, methods of detecting citrus stubborn disease or citrus greening disease, or the presence of the causative agents, Spiroplasma citri, and Candidatus Liberibacter asiaticus, respectively, by detecting one or more secreted protein from the causative agents are provided.

Surprisingly, it has been discovered that the presence of citrus stubborn disease can be detected by detecting a secreted protein as described herein in untreated (unpurified) sap from infected citrus, even trees that are naturally infected and thus have a lower titer of bacteria than an artificially-infected tree. This is particularly surprising as spiralin, a highly-expressed protein from Spiroplasma citri cannot be detected from unpurified sap (see, e.g., FIG. 3B). Accordingly, in some embodiments, the sample of the methods includes fluid of the vascular system (e.g., sap) from a plant located in the field. In some embodiments, a sample is obtained from a plant and is not processed to separate, isolated or purify the secreted protein of interest from other proteins of the sample. For example, the sample is not subjected to extraction, electrophoresis (e.g., polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis), chromatography (e.g., size chromatography, affinity chromatography), or magnetic bead separation prior to detecting the presence of the secreted protein of interest.

Further, in some embodiments, the plant that does not comprise grafting-inoculated citrus tissue or other artificially-inoculated tissue. In some instances, the sample is from phloem-rich tissue.

The methods described herein can be used to detect a secreted protein that is not abundant on S. citri cells. For instance, the secreted protein can be present at a lower level than spiralin which has been shown to be the most abundant protein at the membrane of S. citri cells (see, Duret et al., Appl. Environ. Microbiol., 69:6225-6234 (2003)). Detection of the presence of the secreted protein in a plant sample indicates that the plant has citrus stubborn disease.

In some embodiments, the secreted protein is detected with an antibody. The antibody can recognize (specifically bind) a secreted protein from S. citri or Candidatus Liberbacter, wherein the secreted protein can indicated CSD or HLB, respectively.

Antibody reagents can be used in assays to detect the presence of, or protein expression levels, for the at least one secreted protein in a citrus sample using any of a number of immunoassays known to those skilled in the art. Immunoassay techniques and protocols are generally described in Price and Newman, “Principles and Practice of Immunoassay,” 2nd Edition, Grove's Dictionaries, 1997; and Gosling, “Immunoassays: A Practical Approach,” Oxford University Press, 2000. A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used. See, e.g., Self et al., Curr. Opin. Biotechnol., 7:60-65 (1996). The term immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction with laser induced fluorescence. See, e.g., Schmalzing et al., Electrophoresis, 18:2184-93 (1997); Bao, J. Chromatogr. B. Biomed. Sci., 699:463-80 (1997). Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention. See, e.g., Rongen et al., J. Immunol. Methods, 204:105-133 (1997). In addition, nephelometry assays, in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the protein concentration, are suitable for use in the methods of the present invention. Nephelometry assays are commercially available from Beckman Coulter (Brea, Calif.; Kit #449430) and can be performed using a Behring Nephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biochem., 27:261-276 (1989)).

In some embodiments, the immunoassay includes a membrane-based immunoassay such as an dot blot or slot blot, wherein the biomolecules (e.g., proteins) in the sample are not first separated by electrophoresis. In such an assay, the sample to be detected is directly applied to a membrane (e.g., PVDF membrane, nylon membrane, nitrocellulose membrane, etc). Detailed descriptions of membrane-based immunoblotting are found in, e.g., Gallagher, S R. “Unit 8.3 Protein Blotting:Immunoblotting”, Current Protocols Essential Laboratory Techniques, 4:8.3.1-8.3.36 (2010).

Specific immunological binding of the antibody to the protein of interest can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. An antibody labeled with iodine-125 (¹²⁵I) can be used. A chemiluminescence assay using a chemiluminescent antibody specific for the nucleic acid is suitable for sensitive, non-radioactive detection of protein levels. An antibody labeled with fluorochrome is also suitable. Examples of fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and lissamine. Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, urease, and the like. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p-nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm. An urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals; St. Louis, Mo).

A signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of ¹²⁵I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked antibodies, a quantitative analysis can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, Calif.) in accordance with the manufacturer's instructions. If desired, the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.

The antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), pieces of a solid substrate material or membrane (e.g., plastic, nylon, paper), in the physical form of sticks, sponges, papers, wells, and the like. An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.

Comparative proteomic methods including mass spectrometry (MS) can be used to identify secreted proteins from pathogenic bacteria. For instance, secreted protein profiles from S. citri cells cultures with or without induction of citrus ploem extracts can be determined by MS. Genomic sequencing analysis can be performed to identify gene sequences encoding the secreted proteins. Protein sequence analysis can be used to determine the location of signal peptide cleavage sites based on the amino acid sequence. Secreted proteins for use in the method described herein can be identified using sequence analysis and bioinformatic prediction programs in diseased plants. Expression analysis can also be performed to confirm the presence of the secreted proteins. For instance, the probability of the protein being a Sec-secreted protein can be predicted from a protein's N-terminal secretion signal (e.g., a higher value indicates a greater probability).

Exemplary secreted proteins from Spiroplasma citri whose presence in a citrus sample is indicative of the pathogen (or the corresponding disease citrus stubborn disease) include, the protein encoded by Gene ID: CAK98563 or substantially identical variants. The amino acid sequence of CAK98563 (SEQ ID NO:1) is found in Uniprot No. Q14PL6. Additional secreted proteins from Spiroplasma citri include those described in Table 1 or substantially identical variants thereof. In some embodiments, a protein secreted from S. citri, including any one of those of Table 1, can be used in the method described herein as a detection marker for CSD.

TABLE 1 Secreted proteins from Spiroplasma citri Probability Amino acid SEQ ID of N-terminal position of NO: Protein name secretion signal cleavage site 1 CAK98563 0.777 23 2 CAK99824 0.843 23 3 CAK99227 0.998 29 4 CAK99727 0.906 30 5 CAL0019 0.75 27 6 CAK98956 0.99 29 7 P123-family protein variant A 8 P123-family protein variant B

Exemplary secreted proteins from Candidatus Liberibacter asiaticus whose presence in a citrus sample is indicative of the pathogen (or the corresponding disease citrus greening disease, also known as Huanglongbing or HLB) include those described in Table 2 or substantially identical variants:

TABLE 2 Secreted proteins predicted from Candidatus Liberibacter asiaticus Probabil- MW ity of N- SEQ (kD)of terminal ID mature secretion NO: Protein Name protein Protein function signal 9 CLIBASIA_00460 9 hypothetical protein 0.667 10 CLIBASIA_00995 35 porin outer 0.55 membrane protein 11 CLIBASIA_01135 33 glycine betaine ABC 0.718 transporter 12 CLIBASIA_01295 24 flagellar L-ring 0.637 protein 13 CLIBASIA_01300 17 hypothetical protein 0.546 14 CLIBASIA_01600 35 carboxypeptidase 0.816 15 CLIBASIA_03230 16 hypothetical protein 0.705 16 CLIBASIA_03070 49 pilus assembly 0.723 protein 17 CLIBASIA_02610 45 iron-regulated protein 0.462 18 CLIBASIA_02470 13 putative secreted 0.452 protein 19 CLIBASIA_02425 19 outer membrane 0.884 protein 20 CLIBASIA_02250 20 extracellular solute- 0.55 binding protein 21 CLIBASIA_02145 21 hypothetical protein 0.746 22 CLIBASIA_02120 31 periplasmic solute 0.721 binding protein 23 CLIBASIA_04025 9 hypothetical protein 0.501 24 CLIBASIA_04040 15 hypothetical protein 0.681 25 CLIBASIA_04170 28 rare lipoprotein A 0.56 26 CLIBASIA_04520 33 hypothetical protein 0.603 27 CLIBASIA_04580 10 hypothetical protein 0.795 28 CLIBASIA_05115 17 hypothetical protein 0.664 29 CLIBASIA_05315 14 hypothetical protein 0.706 30 CLIBASIA_00100 15 ABC transporter 0.588 protein 31 CLIBASIA_02075 44 chemotaxis protein 0.624 32 CLIBASIA_03120 4 hypothetical protein 0.65 33 CLIBASIA_04560 19 hypothetical protein 0.57 34 CLIBASIA_05640 5 hypothetical protein 0.668 35 CLIBASIA_05320 7 hypothetical protein 0.83

In some embodiments, a protein secreted from S. citri, including any one of those of Table 1, can be used in the method described herein as a detection marker for CSD.

Also provided are kits, e.g., for use in diagnostic and research applications. The kits can comprise any or all of the reagents to perform the methods described herein. The kits can also comprise a scalpel, razor blade or other implement for obtaining a plant or sap sample from a citrus tree. In addition, the kits can include instructional materials containing directions (i.e., protocols) for the practice of the methods. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

EXAMPLES

This example illustrates a method for serological diagnosis of CSD using a S. citri-specific secreted protein ScCCPP1 which is encoded by CAK98563.

In the study, scCCPP1 was identified using mass spectrometry of the supernatant portion of a bacterial culture of S. citri. The protein was determined to be in relatively high abundance. Using an antibody generated against ScCCPP1 as the antigen, specific signals from S. citri-infected trees were detected using a direct tissue imprint assay. The results demonstrate that this method is suitable for field surveys.

Western blot analysis with rabbit polyclonal anti-ScCCPP1 antibody showed that the secreted pathogen protein was present in the total protein extracts harvested from S. citri cells. ScCCPP1 was also detect uninduced S. citri cells in phloem extracts, as well as induced S. citri cells in either phloem extracts or sucrose only (FIG. 1A). For comparison, spiralin protein which is a major membrane protein of Spiroplasma citri was detected at high levels in the cells including those uninduced and induced (FIG. 1B).

Western blot analysis, as shown in FIG. 2, indicated that ScCCPP1 protein was detected in a protein extract sample from a citrus seedling that was graft-inoculated with Spiroplasma citri (lane 2) or extract samples from CSD-infected adult trees from the field (lane 4-8). Protein extract from S. citri cells (lane 1) was used as a positive control. Protein extract sample from a healthy citrus tree (lane 3) was used as a negative control. The arrowhead of FIG. 2 indicates the position of the predicted MW of ScCCPP1.

In a simple imprint assay, samples of process-free sap from leaves or young branches were obtained from S. citri-infected trees. The plant tissues were removed from the adult trees using a razor blade. The samples were “printed” on a nitrocellulose membrane by pressing the freshly cut cross section of leaf petioles or branches on the membrane, which left faint green-colored marks from the sap in the samples. The membranes were then be taken back to the lab and incubated with a rabbit anti-ScCCPP1 polyclonal antibody for one hour at room temperature (about 22° C.). The membranes were washed with a wash solution to remove any unbound primary antibody. A secondary antibody recognizing rabbit antibodies and conjugated with HRP was added to the membranes and incubated for one hour at room temperature (about 22° C.). The membranes were washed with a wash solution to remove any unbound secondary antibody. Detection was performed using a chemilluminescent reagent and standard autoradiography technique.

Citrus trees that were graft-infected with two S. citri strains (S616 and C189) were examined using an antibody against ScCCPP1 or an antibody against spiralin. ScCCPP1 was detected in imprinted leaf petiole samples from S616-infected trees and C189-infected trees (FIG. 3A). Surprisingly, the spiralin antibody failed to give positive signals from the same samples (FIG. 3B). The results demonstrate that ScCCPP1 can be used as a detection marker for CSD, even in plant samples that do not contain bacterial pathogen cells.

Strong signals of ScCCPP1 were observed in imprints of phloem-rich tissues (e.g., barks) from S. citri-infected citrus trees in the field (FIG. 4A). Four to six different young branches from each tree were printed on the same row of the membrane. Six CSD-infected trees from the field were tested. The arrowhead in FIG. 4A points to a positive signal present exclusively in the regions corresponding to the phloem-rich tissues (e.g., barks) of a branch. This represents the location of the bacterium and the secreted protein. Positive signal was not seen in all tested branches, thus indicating the sporadic distribution of the disease.

Comparison of the imprint method to a nucleic acid-based quantitative PCR (Q-PCR) assay showed that the imprinting immunoassay provides more consistent and more robust detection of CSD infection (FIG. 4B). Young branches from healthy (negative control), graft-inoculated (positive control), and nine field trees were tested by Q-PCR and imprinting immunoassay. Field sample 2 and 5 appeared to be negative for CAK98563 and positive for spiralin by Q-PCR, indicating that the pathogen was absent from these samples. However, the imprint results show that the same samples were actually positive for ScCCPP1 and thus CSD. The data demonstrate that the imprint assay using the ScCCPP1 antibody provides more consistent and robust detection compared to quantitative PCR.

The results also show that the positive signals were mainly present from the regions corresponding to the phloem-rich tissues. This is consistent with the primary location of the antigen. Remarkably, the anti-ScCCPP1 polyclonal antibody gave more reliable and consistent results than an antibody against spiralin protein, which is a major cell membrane component of the bacterial pathogen and one of the most abundant proteins of present in S. citri. These data demonstrate that the presence of CAK98563 indicates citrus stubborn disease in plants even in the absence of S. citri cells. The results also confirm that pathogen secreted proteins can be used as detection markers for phloem-limited bacterial diseases of citrus.

All publications, patents, accession numbers, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. 

What is claimed is:
 1. A kit for detecting citrus stubborn disease or detecting citrus greening disease (Huanglongbing or HLB), the kit comprising one or more reagent specific for a secreted protein from Candidatus Liberibacter asiaticus, wherein the secreted protein consists of SEQ ID NO:29.
 2. The kit of claim 1, wherein the reagent is an antibody.
 3. The kit of claim 2, wherein the antibody is detectably-labeled.
 4. The kit of claim 2, wherein the antibody linked to a solid support.
 5. The kit of claim 1, wherein the reagent is an antibody that specifically binds to a protein consisting of SEQ ID NO:29.
 6. The kit of claim 1, wherein the kit further comprises a secondary antibody that binds to the reagent specific for the secreted protein.
 7. The kit of claim 6, wherein the secondary antibody is labeled with a direct or indirect label.
 8. The kit of claim 6, wherein the label is a direct label and the label is a fluorescent tag, luminescent tag, metal, dye, or radionucleotide linked to the secondary antibody.
 9. The kit of claim 6, wherein the label is an indirect label and the label is an enzyme.
 10. The kit of claim 9, wherein the enzyme is horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, or urease.
 11. The kit of claim 1, further comprising a solid support.
 12. The kit of claim 11, wherein the solid support is a magnetic particle, a chromatographic matrix particle, or the an assay plate.
 13. The kit of claim 12, wherein the assay plate is a microtiter plate.
 14. The kit of claim 1, wherein the reagent specific for a secreted protein is an antibody that specifically binds to a protein comprising SEQ ID NO:29, and the kit further comprises a secondary antibody linked to a direct or indirect label and a solid support.
 15. The kit of claim 14, wherein the secondary antibody that binds to the reagent specific for the secreted protein, wherein the secondary antibody is linked to an indirect label, which is an enzyme.
 16. The kit of claim 14, wherein the solid support is a microtiter plate.
 17. The kit of claim 14, wherein the kit further comprises a positive control.
 18. The kit of claim 14, wherein the kit further comprises a negative control. 